Toxoplasma gondii is a widespread intracellular human pathogen in the same phylum Apicomplexa that includes the human pathogens Plasmodium (the cause of malaria) and Cryptosporidium. Although Toxoplasma is emerging as a model system for the study of intracellular parasitism; our understanding of how host resistance genes control Toxoplasma growth has lagged behind our understanding of how Toxoplasma genes regulate pathogen growth within host cells. A clear understanding of the mechanisms responsible for intracellular host defenses to Toxoplasma will provide useful insights into how this important class of human pathogens can be controlled in infected patients. A major block impeding the functional analysis of how host-cell gene expression controls intracellular Toxoplasma growth is the tack of sensitivity of in vitro assays that measure intracellular parasite growth. To address this issue, a novel FACS-based assay has been developed to measure growth of GFP-tagged Toxoplasma in host cells. This FACS-based assay is significantly more sensitive than previous Toxoplasma growth assays, and can measure the inhibition of parasite growth mediated by individual retroviral-expressed genes in un-stimulated host cells. This new assay permits examination of host-cell responses to Toxoplasma in greater detail than previously possible, and will be used in Aim 1 to determine the mechanism of action of known host resistance genes, particularly the IGTP family of interferon-induced genes whose mechanism of action is unknown, despite having been implicated in the pathogenesis of multiple intracellular pathogens. In proof of principle studies, we have also established that known host resistance genes can be identified in functional screens designed to identify novel host resistance genes. Thus, in Aim 2, functional genetic screens will be conducted using retroviral cDNA libraries to identify host resistance genes that control Toxoplasma infection by (1) active resistance of growth of the virulent tachyzoite form of Toxoplasma, and by (2) passive inter-conversion of the virulent tachyzoite form to the dormant bradyzoite form of Toxoplasma.